Abstract

Lipoxins, which are bioactive lipids derived from ω-6 polyunsaturated fatty acids, play important roles in various biological functions. In this study, the anti-inflammatory effects of lipoxin A4 (LXA4; 5S,6R,15S-trihydroxy-7,9,13-trans-11-eicosatetraenoic acid) were investigated in in vitro cultured cell experiments and in vivo animal experiments. In mouse peritoneal macrophages and mouse macrophage cell line RAW264.7 cells, LXA4 reduced the lipopolysaccharide (LPS)-induced increase in the mRNA expression level of tumor necrosis factor (TNF)-α. LXA4 also reduced the LPS-induced nuclear translocation of nuclear factor-κB (NF-κB). In an LPS-induced acute inflammation mouse model, the injection of LXA4 at 5 μg/kg b.wt. led to down-regulation of the TNF-α level in serum and the TNF-α mRNA expression level in intestinal epithelial cells. Moreover, LXA4 reduced the LPS-caused phosphorylation of IκB kinases, IκB, and NF-κB, the degradation of IκB, and the nuclear translocation of NF-κB in intestinal epithelial cells. In a coculture system using RAW264.7 cells and human colon carcinoma cell line Caco-2 cells, treatment with LXA4 to Caco-2 cells led to reduction of LPS-evoked TNF-α production in RAW264.7 cells and interleukin-8 mRNA expression in Caco-2 cells. These results indicate that LXA4 exerts anti-inflammatory effects through inhibition of NF-κB activation, and, therefore, LXA4 may be useful as a therapeutic strategy against intestinal mucosa inflammation.

Lipoxins, which are composed of lipoxin A4 (LXA4; 5S,6R,15S-trihydroxy-7,9,13-trans-11-eicosatetraenoic acid) and lipoxin B4 (LXB4; 5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid), are bioactive lipids derived from ω-6 polyunsaturated fatty acids. Lipoxins play important roles in various biological functions, especially in inflammatory processes. In contrast to prostaglandins (PGs) and leukotrienes (LTs), which are proinflammatory molecules, lipoxins mainly act in the resolution phase of inflammatory responses and promote the termination of inflammatory processes (Serhan, 2005). In the resolution phase of acute inflammation, “lipid mediator class switching” causes the production of lipoxins (Serhan and Savill, 2005), which then prevent further inflammatory responses by stimulating the uptake and clearance of apoptotic polymorphonuclear neutrophils (PMNs) by macrophages derived from peripheral blood mononuclear cells. Synthesis of lipoxins is induced through cell-cell interactions, for example, the platelet-leukocyte interaction (Serhan and Sheppard, 1990), and lipoxins as well as proinflammatory lipid mediators, i.e., PGs and LTs, are derived enzymatically from arachidonic acid. Lipoxins are formed through 15-lipoxygenase, whereas LTs and PGs are produced through 5-lipoxygenase and cyclooxygenase, respectively. In addition to original lipoxins, it was also reported that acetylsalicylic acid triggered the formation of the carbon-15 position R epimer of LXA4, a more stable analog of LXA4, so-called aspirin-triggered lipoxins (ATLs), or 15-epi-LXA4 (Clària and Serhan, 1995; Serhan, 2005).

Previous studies have reported that LXA4 is able to attenuate airway inflammation (Jin et al., 2007; Levy et al., 2007), and it was also observed that the endogenous LXA4 levels in patients with asthma were decreased (Levy et al., 2005; Planagumá et al., 2008). In addition, LXA4 attenuated the severity of dextran sodium sulfate (DSS)-induced colitis (Gerwirtz et al., 2002). Many researchers have tried to elucidate the molecular mechanisms behind the anti-inflammatory effects of LXA4. LXA4 and ATLs elicit multicellular responses via a specific G protein-coupled receptor termed ALX (Chiang et al., 2006), which was recently designated as ALX/FPR2 in a nomenclature report (Ye et al., 2009). ALX/FPR2 is expressed in PMNs, monocytes, T cells, and macrophages, which are important in the inflammatory process. It was also reported that LXA4, ATLs, and their stable analogs inhibited the neutrophil responses induced by tumor necrosis factor (TNF)-α, one of important proinflammatory cytokines (Hachicha et al., 1999), and there is also a high probability that one of the molecular mechanisms is the suppression of NF-κB activation. LXA4 blocked the activation of NF-κB in pulmonary microvascular endothelial cells (Wu et al., 2008). The activation of NF-κB is the rate-limiting step for various inflammatory responses. The NF-κB family exists in unstimulated cells as homo- or heterodimers bound to IκB family proteins (Sha, 1998). The binding of NF-κB to IκB prevents NF-κB from translocating to the nucleus, thereby maintaining NF-κB in an inactive state. The translocated NF-κB acts as a transcription factor and regulates the expression of various genes encoding proinflammatory cytokines such as TNF-α and IL-12, which have been shown to play important roles in sustained inflammatory responses (Levy et al., 2005, 2007).

In this study, we investigated whether LXA4 exerts anti-inflammatory effects in macrophages. We next examined whether LXA4 was able to reduce inflammatory responses in intestinal epithelial cells, which express ALX/FPR2 (Gronert et al., 1998), using an LPS-induced acute inflammation animal model. Finally, we investigated the anti-inflammatory effects of LXA4 in a coculture system using cultured macrophage RAW264.7 cells and cultured intestinal epithelial Caco-2 cells.

Cell Culture and Treatment.

Mouse macrophage RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium (Wako, Kyoto, Japan) supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. RAW264.7 cells were treated with LXA4 or ethanol as a vehicle control followed by the addition of LPS or saline as a vehicle control for the periods specified in each figure. Human intestinal epithelial Caco-2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 1% MEM-NEAA, 10% fetal calf serum, 4.5 g/l glucose, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. All animal studies were performed according to the Kobe University Animal Experimentation Regulations. To obtain highly purified peritoneal macrophages, 7- to 8-week-old male C57BL/6 mice (Japan CLEA, Shizuoka, Japan) were used. Peritoneal macrophages were harvested by peritoneal lavage administration of phosphate-buffered saline and then were resuspended in RPMI 1640 medium, before being incubated at 37°C in a 5% CO2 humidified incubator. The adherent cells were used to perform the experiments. Peritoneal macrophages were treated with LXA4 or ethanol as a vehicle control and then LPS or saline as a vehicle control for the periods specified in each figure.

Coculture System.

The coculture system was constructed according to the method of a previous report (Tanoue et al., 2008). In brief, Caco-2 cells were seeded at 3.75 × 105 cells/well in Transwell insert plates (4.67 cm2, 0.4-μm pore size; Corning Costar, Cambridge, MA). The cells were used in passage numbers 48 to 62. The cell culture medium was changed every 3 days for 21 days until the cells had fully differentiated, and the transepithelial electrical resistance value was measured using a Millicell-ERS instrument (Millipore Corporation, Billerica, MA). After confirmation of differentiation by the transepithelial electrical resistance value (higher than 350 ohm × cm2), Caco-2 cells were subjected to the following experiment. RAW264.7 cells were seeded at 8.5 × 105 cells/well in six-well tissue culture plates and incubated overnight so that they completely adhered to the well, and the cells were used in passage numbers 10 to 30. After all media was replaced with RPMI 1640, the Transwell inserts on which the Caco-2 cells had been cultured were added into multiple-plate wells preloaded with RAW264.7 cells. To evaluate the anti-inflammatory effects of LXA4 in the coculture system, LXA4 at various concentrations was applied to the apical side. After 3 h, 1 ng/ml LPS was added to the basolateral side. After an additional incubation for 3 h, the culture supernatant from the basolateral side was collected to measure the TNF-α level. The treated cells were harvested to isolate total RNA, which then was subjected to real-time polymerase chain reaction (PCR).

Animal Experiment.

Seven- to 8-week-old male C57BL/6 mice received intraperitoneal injections of 5 μg/kg b.wt. LXA4 or corn oil as a vehicle control. The dosage amount of LXA4 was decided according to the report by Menezes-de-Lima et al. (2006). After 30 min, the mice received intraperitoneal injections of 250 μg/kg b.wt. LPS or saline as a vehicle control. The mice were sacrificed 1 h after LPS stimulation, and their blood was collected via cardiac puncture. Twenty-four hours after LPS administration, the intestinal epithelial cells from the entire mucosal layer of small intestine were also obtained.

Total RNA of RAW264.7 cells, peritoneal macrophages, and intestinal epithelial cells was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription (RT) was performed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Tokyo, Japan) according to the manufacturer's instructions. The PCR was performed using the Blend Taq System (TOYOBO, Osaka, Japan). PCR reactions were performed in 50 μl of reaction mixture containing 5 μl of 10× buffer for Blend Taq, 5 μl of 2 mM deoxynucleotide triphosphate, 1 μl of 10 μM sense primer, 1 μl of 10 μM antisense primer, 0.5 μl of 2.5 units/μl Blend Taq, 33.5 μl of distilled water, and 4 μl of 0.25 μg/μl cDNA solution. The amplification protocols for ALX/FPR2 and β-actin consisted of predenaturing at 94°C for 5 min, 30 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, and final extension at 72°C for 7 min. The following primers were used: mouse ALX/FPR2, 5′-CCTTGGCTTTCTTCAACAGC-3′ and 5′-GCACAGTGGAACTCAAAGCA-3′; and β-actin, 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3′. Amplified cDNA was separated by 2% agarose gel electrophoresis and visualized with ethidium bromide.

Quantitative Real-Time Polymerase Chain Reaction.

Quantitative real-time PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocols. The reaction conditions were 40 cycles of two-stage PCR consisting of denaturation at 95°C for 15 s and annealing at 60°C for 1 min after an initial denaturation step at 95°C for 10 min. The primer sequences were as follows: mouse TNF-α, 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ and 5′-TGGGAGTAGACAAGGTACAACCC-3′; IL-12, 5′-AGCAGTAGCAGTTCCCCTGA-3′ and 5′-AGTCCCTTTGGTCCAGTGTG-3′; and β-actin, 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3′. For the relative comparison of mRNA expression levels, the data from real-time PCR were analyzed with a ΔΔCt method and normalized to the amount of β-actin cDNA as an endogenous control.

Enzyme-Linked Immunosorbent Assay.

The culture medium was centrifuged at 16,000g for 5 min at 4°C, and the supernatant was collected. The mouse serum was separated from the blood by centrifugation at 15,000g for 10 min at 4°C. The TNF-α levels in the culture medium and serum were measured by ELISA (eBioscience, San Diego, CA) according to the manufacturer's protocol.

Western Blot Analysis.

Peritoneal macrophages and intestinal epithelial cells were homogenized in lysis buffer A (10 mM Tris-HCl, 10 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1.0 mM dithiothreitol). The cell lysate was incubated on ice for 10 min and centrifuged at 1600g for 10 min at 4°C. The supernatant obtained was used as a source of postnuclear proteins for Western blot analysis. The precipitate was resuspended in lysis buffer A, incubated on ice with 10 min, and centrifuged at 1600g for 15 min at 4°C. After this process was repeated three times, the precipitate obtained was suspended in 1 ml of lysis buffer A without Triton X-100. The homogenates were vortexed for 30 s and then centrifuged at 1600g for 10 min at 4°C. The precipitate was resuspended in lysis buffer B (10 mM Tris-HCl, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1.0 mM dithiothreitol) and incubated on ice for 30 min with occasional mixing. The homogenates were centrifuged at 15,000g for 20 min at 4°C, and the supernatant obtained was used as a source of nuclear proteins for Western blot analysis.

The proteins were boiled in a quarter volume of sample buffer (1 M Tris-HCl, pH 7.5, 640 mM 2-mercaptoethanol, 0.2% bromphenol blue, 4% SDS, and 20% glycerol) and were separated on 10% SDS-polyacrylamide gels. The proteins on the gels were transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 1% skim milk in TBS-T (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.5% Tween 20) and was probed with each primary antibody before being reacted with the corresponding horseradish peroxidase-conjugated secondary antibody. The protein-antibody complex was visualized with ChemiLumi ONE (Nacalai Tesque, Kyoto, Japan) and was detected using an Image Reader (LAS-3000 Imaging System; Fuji Photo Film, Tokyo, Japan).

L929 Cytotoxicity Assay.

The TNF-α level in the Caco-2/RAW264.7 coculture system was quantified using a cytotoxicity assay with L929 cells, which are an actinomycin D-treated murine fibroblast cell line. In this experiment, murine TNF-α was used as the standard according to the method of the report by Takada et al. (1994).

Statistical Analysis.

All data are expressed as means ± S.E. of at least three independent determinations for each experiment. Statistical significance was analyzed using Student's t test, and a level of probability of 0.05 was used as the criterion of significance.

Results

LXA4 Reduces the Expression of Cytokines and the Activation of NF-κB in Peritoneal Macrophages.

To investigate the anti-inflammatory effects of LXA4 in macrophages, we used peritoneal macrophages derived from C57BL/6 mice. First we examined the expression of the LXA4 receptor, ALX/FPR2 (Fig. 1A), and the expression of ALX/FPR2 mRNA in murine peritoneal macrophages was confirmed by RT-PCR. Next, we investigated whether LXA4 was able to reduce LPS-induced expression of TNF-α and IL-12, which play important roles in inflammatory responses (Rogler and Andus, 1998) (Fig. 1, B and C). Treatment with 100 ng/ml LPS increased the TNF-α mRNA expression level 350-fold (100%), and 300 nM LXA4 decreased the induced level by 62%. LPS (100 ng/ml) also up-regulated IL-12 mRNA expression level 500-fold, and 300 nM LXA4 significantly reduced LPS-induced IL-12 expression. Conversely, 100 nM LXA4 could not significantly reduce LPS-caused expressions of TNF-α and IL-12 (data not shown).

LXA4 reduces TNF-α and IL-12 production in peritoneal macrophages. A, expression of ALX/FPR2 mRNA in peritoneal macrophages. Total RNA was isolated from peritoneal macrophages, and the mRNA expression levels of ALX/FPR2 and β-actin were determined by RT-PCR as described under Materials and Methods. B, reduction of LPS-induced TNF-α mRNA expression by LXA4 in peritoneal macrophages. Peritoneal macrophages were pretreated with 300 nM LXA4 or ethanol as a vehicle control for 4 h and then stimulated with 100 ng/ml LPS or saline as a vehicle control for 3 h. The mRNA expression level of TNF-α was examined by quantitative real-time PCR as described under Materials and Methods. The mRNA expression levels were normalized to those of β-actin as an internal standard, and data are presented as the mean ± S.E. (n = 3). *, significant differences according to a Student's t test (p < 0.05). C, reduction of LPS-induced IL-12 mRNA expression by LXA4 in peritoneal macrophages. The IL-12 mRNA expression level was examined by quantitative real time PCR as described under Materials and Methods. mRNA expression levels were normalized to those of β-actin as an internal standard, and data are presented as the mean ± S.E. (n = 3). *, significant differences according to a Student's t test (p < 0.05). D, inhibition of LPS-induced NF-κB p65 nuclear translocation by LXA4 in peritoneal macrophages. Peritoneal macrophages were pretreated with 300 nM LXA4 or ethanol as a vehicle control for 4 h and then stimulated with 100 ng/ml LPS or saline as a vehicle control for 30 min. The NF-κB proteins in nucleus were detected by Western blotting as described under Materials and Methods. Typical images are shown from at least triplicate determinations. bp, base pairs.

LXA4 Reduces the Expression of TNF-α and the Activation of NF-κB in RAW264.7 Cells.

To confirm whether LXA4 can reduce TNF-α expression in other types of cells, we selected the mouse macrophage cell line RAW264.7 cells because the expression of ALX/FPR2 in RAW264.7 cells was also observed (Fig. 2A). Treatment with 100 ng/ml LPS to RAW264.7 cells led to a significant increase in the TNF-α mRNA expression level, and 300 nM LXA4 reduced its expression level to 55% (Fig. 2B). LXA4 at the concentration of 300 nM also decreased 100 ng/ml LPS-induced TNF-α production significantly (Fig. 2C). Moreover, in an immunostaining method, we found that 100 ng/ml LPS caused the nuclear translocation of NF-κB, and pretreatment with 300 nM LXA4 inhibited the LPS-evoked NF-κB nuclear translocation (Fig. 2D). These results suggest that LXA4 exerts anti-inflammatory effects through the inhibition of NF-κB activation.

LXA4 reduces TNF-α production in RAW264.7 cells. A, expression of ALX/FPR2 mRNA in RAW264.7 cells. Total RNA was isolated from RAW264.7 cells, and the mRNA expression levels of ALX/FPR2 and β-actin were determined by RT-PCR as described under Materials and Methods. B, reduction of LPS-induced TNF-α mRNA expression by LXA4 in RAW264.7 cells. RAW264.7 cells were pretreated with 300 nM LXA4 or ethanol as a vehicle control for 4 h and then stimulated with 200 ng/ml LPS or saline as a vehicle control for 6 h. The mRNA expression levels of TNF-α and IL-12 were examined by quantitative real time PCR as described under Materials and Methods. mRNA expression levels were normalized to those of β-actin as an internal standard, and data are presented as the mean ± S.E. (n = 3). *, significant differences according to a Student's t test (p < 0.05). C, reduction of LPS-induced TNF-α production by LXA4 in RAW264.7 cells. RAW264.7 cells were treated with 300 nM LXA4 or ethanol as a vehicle control for 4 h followed by stimulation with 200 ng/ml LPS or saline as a vehicle control for 24 h. The TNF-α level in the culture medium was measured by ELISA as described under Materials and Methods. Data are presented as the mean ± S.E. (n = 3). *, significant differences according to a Student's t test (p < 0.05). D, inhibition of LPS-induced NF-κB p65 nuclear translocation by LXA4 in RAW264.7 cells. RAW264.7 cells were pretreated with 300 nM LXA4 or ethanol as a vehicle control for 12 h and then incubated with 200 ng/ml LPS or saline as a vehicle control for 1 h. The cells were subjected to an immunofluorescent staining method with anti-NF-κB p65 antibody as described under Materials and Methods (white: NF-κB). bp, base pairs.

LXA4 Exerts Anti-Inflammatory Effects in Vivo.

We induced acute systemic inflammation via an intraperitoneal injection of LPS and examined whether the anti-inflammatory effects of LXA4 could be observed in vivo. The intraperitoneal injection of 250 μg/kg b.wt. LPS significantly increased the serum TNF-α level (to approximately 5 ng/ml), and pretreatment with 5 μg/kg b.wt. LXA4 decreased the TNF-α level to 1.9 ng/ml (Fig. 3A). Next, we investigated the TNF-α mRNA expression level in intestinal epithelial cells 24 h after LPS injection (Fig. 3B). LPS injection led to a significant increase in the TNF-α mRNA expression level, and pretreatment with 5 μg/kg b.wt. LXA4 reduced the TNF-α level to 38%. Next, the influence of LXA4 in the NF-κB signaling pathway was examined (Fig. 3C). LXA4 could inhibit the LPS-evoked nuclear translocation of NF-κB and its phosphorylation. The LPS-caused degradation and phosphorylation of IκB were also reduced by LXA4. In addition, LXA4 inhibited the IKKα/β phosphorylation evoked by LPS. These results suggest that LXA4 can reduce NF-κB activation through the inhibition of IKKα/β.

LXA4 reduces intestinal inflammation in vivo. A, reduction of LPS-induced TNF-α production by LXA4. C57BL/6 mice received intraperitoneal injections of 5 μg/kg b.wt. (BW) LXA4 or corn oil as a vehicle control. After 30 min, the mice received intraperitoneal injections of 250 μg/kg b.wt. LXA4 or saline as a vehicle control. One hour after LPS administration, the serum TNF-α level was measured by ELISA as described under Materials and Methods. Data are presented as the mean ± S.E. (n = 3). *, significant differences according to a Student's t test (p < 0.05). B, reduction of LPS-induced TNF-α mRNA expression by LXA4 in murine intestinal epithelial cells. The mice were treated with LXA4 and LPS as described in the legend to Fig. 3A. Twenty-four hours after LPS administration, the TNF-α mRNA expression level in intestinal epithelial cells was examined by quantitative real-time PCR as described under Materials and Methods and was normalized to that of β-actin as an internal standard. Data are presented as the mean ± S.E. (n = 3). *, indicate significant differences according to a Student's t test (p < 0.05). C, regulation of the NF-κB signaling by LXA4 in murine intestinal epithelial cells. The mice were treated with LXA4 and LPS as described in the legend to Fig. 3A. Twenty-four hours after LPS administration, the NF-κB, phosphorylated NF-κB, IκB, phosphorylated IκB, IKKα, IKKβ, and phosphorylated IKKα/β proteins in intestinal epithelial cells were detected by Western blotting as described under Materials and Methods. Typical images are shown from at least triplicate determinations.

LXA4 Reduces the Expression of Cytokines in a Coculture System Using Caco-2 and RAW 264.7 Cells.

Finally, an in vitro coculture system was used to examine the anti-inflammatory effects of LXA4 via the direct interaction of LXA4 with intestinal cells. A coculture model using a combination of intestinal epithelial Caco-2 cells and macrophage RAW264.7 cells was developed as an in vitro intestine model, and LPS was applied to the basolateral side to imitate the gut inflammatory process. LXA4 at concentrations of 1, 50, and 100 nM was added to the apical side of the coculture system followed by treatment with 1 ng/ml LPS to the basolateral side. Treatment with LPS to the basolateral RAW264.7 cells led to significant up-regulation of the IL-8 mRNA levels in the apical Caco-2 cells, and LXA4 dose dependently decreased the LPS-evoked enhancement of the IL-8 mRNA level in Caco-2 cells (Fig. 4A). In addition, LXA4 at the concentrations of 50 and 100 nM significantly reduced the TNF-α production induced by LPS in RAW264.7 cells (Fig. 4B).

Anti-inflammatory effects of LXA4 in the coculture system. A, reduction of IL-8 mRNA expression by LXA4 in Caco-2 cells. LXA4 at 1, 50, and 100 nM was added to the apical compartment of the Caco-2/RAW264.7 coculture model. After 3 h, 1 ng/ml LPS was added to the basolateral compartment, followed by incubation for an additional 3 h. IL-8 mRNA expression in Caco-2 cells was detected by real-time PCR as described under Materials and Methods and was normalized to that of β-actin as an internal standard. Data are represented as the mean ± S.E. (n = 3). *, significant differences according to the Student's t test (p < 0.05). B, reduction of TNF-α production by LXA4 in RAW264.7 cells. TNF-α production in RAW264.7 cells was determined by a L929 cytotoxicity assay as described under Materials and Methods. Data are represented as the mean ± S.E. (n = 3). *, significant differences according to a Student's t test (p < 0.05).

Discussion

Lipoxins, bioactive lipids derived from ω-6 polyunsaturated fatty acids, play important roles in inflammatory processes. Many researchers have investigated the bioactive functions of lipids derived from polyunsaturated fatty acids. For example, resolvin E1, an endogenous lipid mediator derived from eicosapentaenoic acid, protected against colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) (Arita et al., 2005) and DSS (Ishida et al., 2009). In this study, we investigated whether LXA4, one of the lipoxins, was able to reduce inflammatory responses in in vitro experiments using peritoneal macrophages and mouse macrophage cell line RAW264.7 cells and in in vivo experiments using an acute inflammatory mouse model. In addition, the anti-inflammatory effects of LXA4 were examined in an in vitro coculture system constructed with macrophages and intestinal epithelial cells. LXA4 was originally found as an anti-inflammatory and proresolutionary eicosanoid, and the anti-inflammatory effects of LXA4 have been observed in various types of inflammatory diseases including asthma (Levy et al., 2002), dermal inflammation (Guilford et al., 2004), and arthritis (Fiore et al., 2005). It was also reported that the oral administration of an LXA4 analog attenuated DSS-induced colitis (Gewirtz et al., 2002) and that the oral administration of another LXA4 analog also had anti-inflammatory effects on a TNBS-induced colitis model (Fiorucci et al., 2004). In this study, LXA4 was able to reduce LPS-evoked inflammatory responses in peritoneal macrophages from C57BL/6 mice (Fig. 1, B and C) and mouse macrophage cell line RAW264.7 cells (Fig. 2, B and C). Moreover, the intraperitoneal administration of LXA4 attenuated the intestinal inflammatory responses induced by the intraperitoneal injection of LPS (Fig. 3, A and B). In addition, treatment with LXA4 to the intestinal cells also led to reduction of the inflammatory responses evoked by LPS in a coculture system using intestinal cells and macrophages as shown in Fig. 4, A and B. Inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease are characterized by chronic inflammation of the intestinal tract. Although the precise mechanisms of their pathogenesis are still unknown, it has been suggested that IBD results from an aberrant innate immune response to normally nonpathogenic gut flora. It was reported that intestinal epithelial cells and macrophages in the inflamed intestines of patients with IBD secreted large amounts of proinflammatory cytokines (Arai et al., 1998), and activated NF-κB was also detectable in biopsy specimens of patients with IBD (Schreiber et al., 1998). These results indicate that IBD is caused by not only direct inflammatory stimulation of the intestinal cells but also indirect inflammatory stimulation via macrophages. In the intestinal epithelial cells, the expression level of the LXA4 receptor is up-regulated by the inflammatory stimuli (Gronert et al., 2002). In addition, the expression of the LXA4 receptor in macrophages was confirmed (Fig. 1A). Therefore, LXA4 may be effective against various intestinal diseases including IBD.

The anti-inflammatory effects of LXA4 may be dependent on suppression of NF-κB activation. NF-κB activation is the rate-controlling step for inflammatory processes. Regarding bowel inflammation, an increase in the nuclear NF-κB p65 level was observed in TNBS-induced colitis in mice, and the intravenous injection of the NF-κB p65 antisense oligonucleotide (an oligonucleotide designed to target the translation initiation site of murine NF-κB p65) attenuated TNBS caused-colitis (Neurath et al., 1996). Moreover, a significant up-regulation of the NF-κB p65 level in the nuclei of macrophages and endothelial cells from patients with Crohn's disease was found (Neurath et al., 1996). These results suggest that NF-κB activation is one of the main mechanisms behind intestinal inflammation including IBD, and there is a possibility that reduced NF-κB activation can contribute to the treatment of colitis.

Destruction of the mucosal barrier caused by exogenous antigens leads to exposure of the lamina propria to luminal bacterial antigens, which, in turn, recruit and activate innate immune cells including macrophages (Tlaskalová-Hogenová et al., 2005). It was also reported that the elevated level of macrophages in the colonic mucosa is associated with the severity of DSS-induced colitis in mice (Ohkawara et al., 2008). Macrophages play important roles during inflammatory responses. LPS stimulates Toll-like receptor 4 on the surfaces of macrophages, which triggers the subsequent activations of the downstream signaling pathways; the main signaling pathway is NF-κB signaling. In this study, LXA4 was able to inhibit the LPS-caused nuclear translocation of NF-κB in peritoneal macrophages (Fig. 1D) and mouse macrophage RAW264.7 cells (Fig. 2D). In addition, the intestinal inflammatory responses to an intraperitoneal injection of LPS were attenuated by the intraperitoneal injection of LXA4 (Fig. 3, A and B). LXA4 also inhibited the LPS-evoked phosphorylation of IKKs, IκB, and NF-κB, the degradation of IκB, and the nuclear translocation of NF-κB (Fig. 3C). It was also reported that lipoxins reduced the nuclear translocation of NF-κB in human PMNs and monocytes (József et al., 2002). These results indicate that LXA4 exerted anti-inflammatory effects in immune cells through the inhibition of NF-κB activation, resulting in reduction of bowel inflammation responses and a decrease in the damage caused to the intestinal epithelium. Moreover, treatment with LXA4 to intestinal epithelial cells in the coculture system using intestinal epithelial cell line Caco-2 cells and macrophage cell line RAW264.7 cells led to reduction of the IL-8 level in Caco-2 cells and a decrease in TNF-α production in RAW264.7 cells (Fig. 4, A and B). In the coculture system, LXA4 and LPS were applied to Caco-2 cells and RAW264.7 cells, respectively. There is a high probability that the direct action of LXA4 of Caco-2 cells led to the remarkable decreased expression of IL-8 in Caco-2 cells. The indirect action of LXA4 on RAW264.7 cells via Caco-2 cells might also cause moderately decreased expression of TNF-α in RAW264.7 cells treated with LPS. In a previous study, the expression of ALX/FPR2 was observed in human enterocytes (Gronert et al., 2002). Moreover, LXA4 reduced the Salmonella typhimurium-evoked secretion of IL-8 in T84 intestinal epithelial cells (Gewirtz et al., 1998). Therefore, from our results, there is the possibility that the actions of LXA4 on intestinal epithelial cells occur via ALX/FPR2 followed by signal transduction to the innate immune cells (including macrophages) and the subsequent reduction in inflammatory responses.

Our finding indicates that LXA4 can regulate the NF-κB signaling pathway. In this study, LXA4 could inhibit the phosphorylation of IKKs and the subsequent NF-κB signaling (Fig. 3C). A previous study suggested regulation of the NF-κB signaling pathway by LXA4 and ATLs (Fiore et al., 2005), and it was also reported that ATLs reduced the NF-κB-mediated transcriptional activation in an ALX-dependent manner and inhibited the degradation of IκBα (Gewirtz et al., 2002). Therefore, induction of anti-inflammatory responses by LXA4 may be dependent on the NF-κB signaling pathway, although the ALX-dependent mitogen-activated protein kinase signaling pathway has been proposed (Ohira et al., 2004).

In conclusion, LXA4 is able to attenuate the inflammatory responses in intestinal cells through the inhibition of NF-κB activation, and the anti-inflammatory effects of LXA4 in the intestine may be dependent on not only direct action of LXA4 to intestinal epithelial cells but also indirect action via immune cells including macrophages. The in vivo experiments also revealed that the intraperitoneal injection of 5 μg/kg b.wt. LXA4 reduced the LPS-caused inflammatory responses in intestinal cells (Fig. 3). In general, anti-inflammatory agents require a relatively high dosage for the treatment. For example, the intraperitoneal injection of budesonide, one of the therapeutic agents against Crohn's disease, exerted anti-inflammatory effects in the mouse colitis model using 2,4-dinitrobenzenesulfonic acid, and the effective dosage was 431 μg/kg b.wt. (Dahlbäck et al., 1986). Therefore, the reduction of LPS-caused inflammatory responses by 5 μg/kg b.wt. LXA4 is meaningful, and these results may offer a promising therapeutic strategy against intestinal diseases including IBD.

Footnotes

This study was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Global Center of Excellence for Education and Research on Signal Transduction Medicine in the Coming Generation Grant F301].

(2003) Stimulation of Toll-like receptor 4 by lipopolysaccharide during cellular invasion by live Salmonella typhimurium is a critical but not exclusive event leading to macrophage responses. J Immunol170:5445–5454.